DNA polymerases are essential for maintaining genomic order during DNA replication and repair and thus for the long-term survival of a species. When DNA damage arising from a variety of exogenous and endogenous sources (e.g. environmental chemicals and radiation, smoking, thermal aberrations) is not accurately repaired, it can lead to human diseases like colon, lung, or skin cancer and premature aging. Thus, understanding polymerase fidelity mechanisms in DNA synthesis represents a fundamental biological and biomedical challenge. The fidelity of DNA polymerases broadly refers to their ability to incorporate correct rather than incorrect nucleotides complementary to the template DNA; such fidelities span a wide range, from 1 to nearly 10(E6) errors per one million nucleotides incorporated. Based on extensive structural and kinetic data as well as theoretical studies for several DNA polymerases, we hypothesize that high fidelity enzymes tightly orchestrate the assembly of the active site prior to nucleotide incorporation, while lower fidelity polymerases have a more flexible active site and thus a distinct assembly process; characteristic differences in the electrostatic environment and plasticity of the binding pocket likely result. Since static crystallographic structures and kinetic experimental studies of DNA polymerases cannot describe complete dynamic and energetic effects of the active site, dynamics simulations are well poised, and critically needed, to complement polymerase experimental results. In our collaborative project between an experimental and theoretical team, we will investigate systematically at atomic resolution how the conformational changes and nucleotide incorporation (chemical) pathways for higher-fidelity (pol beta) and low-fidelity (Dpo4) polymerases dictate different steering mechanisms, and how the template base, incoming nucleotide, key protein residues, and lesion-modified DNA affect the binding pocket electrostatic environment/plasticity and thus fidelity. These aims will be achieved by a combination of long-time molecular dynamics simulations and novel methodologies (transition path sampling, stochastic path approach, principal component analysis, and mixed quantum-classical mechanics methods) and an iterative design between theory and experimentation for testing, validating, and expanding these hypotheses. In particular, by delineating complete reaction profiles (conformational change and chemistry) for correct and incorrect basepairs in pol beta and relating them to experimentally-determined catalytic efficiencies and fidelity values, we will propose the rate-limiting step, orchestration of the active site assembly, and fidelity mechanisms involved and subsequently test them by experiments on mutant systems. Moreover, we will test our hypothesis that subtle conformational changes in Dpo4's thumb and little finger domains are closely associated with Dpo4's low-fidelity and lesion bypassing mechanisms, which are likely distinct than pol beta's. Our long term goals are to bridge macroscopic polymerase structures and kinetic measurements regarding catalytic efficiency, fidelity, and nucleotide binding affinity to better understand fidelity mechanisms of DNA polymerases, including response to oxidative damage and other lesions. Such studies have immediate applications to the diagnostics, and eventually treatment via polymerase inhibitors, of human diseases caused by defective repair of DNA, like various cancers and premature aging.